Dengue virus (DENV) is an arthropod-borne virus that is endemic in tropical and subtropical regions throughout the world and is classified by the NIAID as a Category A viral pathogen of biodefense. The global incidence of DENV infection, including that in the US, has increased dramatically in recent decades. Infection with DENV often results in serious disease, such as hemorrhagic fever which has a fatality rate of up to 5%. Currently, no vaccines or specific antiviral therapy are available against DENV infection. DENV is a flavivirus that employs a unique molecular mechanism for the initiation of viral protein translation which is regulated by the 5' and 3' untranslated regions (UTR) of the viral RNA. This application is aimed at exploiting DENV translation as a target for antiviral chemotherapy. The objective of this application is to identify and develop a small molecule that inhibits DENV replication and meets the criteria to be a Development Candidate for further preclinical evaluation within the 5-year research period proposed. The ultimate goal of this project is to develop an antiviral agent that will be approved for the therapy of DENV infection. A stably transfected clonal 293H cell line containing a DENV UTR-mediated translation reporter construct has been established and employed in the performance of a high throughput screening of PTC's small molecule library. In a high throughput assay, hit compounds were identified that selectively inhibit the DENV UTR mediated translation of a luciferase reporter. Selected hits also inhibit DENV replication in cell culture. This application is to further characterize and optimize these hit compounds towards the identification of a Development Candidate. There are four Specific Aims comprising this application. Specific Aim 1 is to further characterize the hit compounds toward identifying lead compounds. Specific Aim 2 is directed towards lead optimization of selected compounds to improve their potency and pharmaceutical properties in order to identify a Development Candidate suitable for clinical trials in DENV infected patients. An ideal Development Candidate will be active against all four serotypes of DENV, have good selectivity with respect to cytotoxicity and mammalian cap-dependent protein translation, and have optimal pharmaceutical and safety profiles. Specific Aim 3 will be to conduct preclinical IND-enabling toxicology and safety pharmacology studies of a DC in support of IND application. Finally, Specific Aim 4 will be to conduct drug resistance and mechanism of action studies to demonstrate target engagement and to characterize the molecular mechanism of action of selected DENV inhibitors. Successful completion of this project will result in the discovery and development of molecules with clinical utility for the therapy of DENV infection, a worldwide and urgent medical need.